Electrophoresis methods

ABSTRACT

A separation carrier comprising β-1,3-glucan and/or methyl cellulose; a running buffer comprising the separation carrier; a method of capillary electrophoresis or microchip electrophoresis, wherein a sample comprising macromolecular compounds is run in the presence of the running buffer; a method of capillary electrophoresis or microchip electrophoresis, comprising the step of injecting the sample by an electrical injection or application of pressure; and a method for analyzing macromolecular compounds by the method of capillary electrophoresis or microchip electrophoresis. According to the present invention, it is possible to achieve high resolution quickly. Therefore, the methods are useful in the High Through-put screening analysis of proteins or sugar chains in gene analysis, proteome analysis or glycome analysis, and applicable in medical diagnostic apparatuses and the elucidation of biological functions, mechanisms of onset of diseases, etc.

TECHNICAL FIELD

[0001] The present invention relates to a method of electrophoresis anda method for analyzing macromolecular compounds, which are suitable forthe electrophoresis of macromolecular compounds and by which highresolution can be achieved conveniently and quickly; and are applicableto gene analysis, proteome analysis, glycome analysis and the like. Morespecifically, the present invention relates to a separation carrier, arunning buffer, a method of electrophoresis and a method for analyzingmacromolecular compounds, by which macromolecular compounds such asproteins, peptides, amino acids, sugar chains, polysaccharides andnucleic acids (e.g., DNA, RNA) can be separated quickly at highresolution.

BACKGROUND ART

[0002] With human genome analysis, it has been expected that genomefunctions will be elucidated. Specifically, it is expected that bytranscriptome analysis, proteome analysis, metabolome analysis, glycomeanalysis, etc., for example, expression of transcription products basedon nucleotide sequence information and functions of gene products(proteins), in vivo metabolites, sugar chains, etc., will be elucidatedto thereby elucidate the mechanisms of onset of diseases. Additionally,by the elucidation of the mechanisms of onset of diseases, applicationsto the prevention, treatment, etc. of such diseases are expected.

[0003] Currently, regarding proteome analysis, for example, profiling ofprotein by two-dimensional electrophoresis, etc. are conducted. However,the aforementioned two-dimensional electrophoresis has drawbacks suchthat the procedures are complicated, that long operating time isrequired, and that large volume of sample is required.

[0004] Additionally, in some cases, capillary electrophoresis andmicrochip electrophoresis may be used to analyze proteins or sugarchains. However, when analyzing a protein by the capillaryelectrophoresis or microchip electrophoresis, since the protein may beadsorbed in some cases to the capillary. Therefore, there is a drawbackthat the development thereof may be interfered, which makes it difficultto analyze it.

[0005] Furthermore, since conventional electrophoresis has a limitationas to shortening the separation time in order to achieve high-speedseparation, electrophoresis is conducted under high voltage. However,there is a drawback of reduced resolution due to the high voltageexerted in some cases.

[0006] Accordingly, there is a need for the establishment of techniquesfor High Through-put screening analysis of macromolecular compounds,specifically, proteins and sugar chains, of which operations are simpleand by which high resolution can be obtained quickly.

DISCLOSURE OF INVENTION

[0007] An object of the present invention is to provide a separationcarrier, a running buffer and a method of electrophoresis, capable ofanalyzing macromolecular compounds such as proteins, peptides, aminoacids, sugar chains, polysaccharides and nucleic acids (e.g., DNA, RNA,etc.), which are suitable for the electrophoresis of macromolecularcompounds, and of which operations are simple and by which highresolution can be obtained quickly, as well as a method for analyzingsuch macromolecular compounds based on the method of electrophoresis.

[0008] Specifically, the gist of the present invention relates to:

[0009] [1] a separation carrier usable for capillary electrophoresis ormicrochip electrophoresis, wherein the separation carrier comprises onekind of compound selected from the group consisting of β-glucan andmethyl cellulose;

[0010] [2] the separation carrier described in item [1] above, whereinthe separation carrier comprises as the β-glucan at least one kindselected from the group consisting of laminaran containing β-1,3-glucan,curdlan containing β-1,3-glucan, a plant extract containingβ-1,3-glucan, a seaweed extract containing β-1,3,3-glucan, an yeastextract containing β-1,3,3-glucan, a fungal extract containingβ-1,3-glucan, and a fungal cultured medium containing β-1,3-glucan;

[0011] [3] the separation carrier described in item [1] above, whereinthe separation carrier comprises a seaweed extract containingβ-1,3-glucan as the β-glucan;

[0012] [4] the separation carrier described in item [3] above, whereinthe seaweed extract is an extract obtained by subjecting raw materialseaweed to one kind of extraction method selected from the groupconsisting of a water extraction, an acid/alkali extraction and asolvent extraction;

[0013] [5] a running buffer usable for capillary electrophoresis ormicrochip electrophoresis, wherein the running buffer comprises theseparation carrier of any one of items [1] to [4] above;

[0014] [6] the running buffer described in item [5] above, wherein therunning buffer is one kind of buffer selected from the group consistingof the following (1) to (3):

[0015] (1) a buffer comprising a phosphate buffer at pH 1.0 to 12.0 at aconcentration of 1 mM to 0.5 M;

[0016] (2) a buffer comprising a borate buffer at pH 5.0 to 11.0 at aconcentration of 1 mM to 0.5 M; and

[0017] (3) a buffer comprising a Tris-borate buffer at pH 5.0 to 11.0 ata concentration of 1 mM to 0.5 M, and wherein the running buffercomprises methyl cellulose at a concentration of 0.001 to 0.5% byweight;

[0018] [7] the running buffer described in item [5] above, wherein therunning buffer is one kind of buffer selected from the group consistingof the following (1) to (4):

[0019] (1) a buffer comprising a phosphate buffer at pH 1.0 to 12.0 at aconcentration of 1 mM to 0.5 M;

[0020] (2) a buffer comprising a borate buffer at pH 5.0 to 11.0 at aconcentration of 1 mM to 0.5 M;

[0021] (3) a buffer comprising a Tris-borate buffer at pH 5.0 to 11.0 ata concentration of 1 mM to 0.5 M; and

[0022] (4) a buffer further comprising 0.001 to 1.0% by weight of methylcellulose in the buffer of the above item (3), and wherein the runningbuffer comprises curdlan at a concentration of 0.000001 to 0.1% byweight;

[0023] [8] the running buffer described in item [5] above, wherein therunning buffer is one kind of buffer selected from the group consistingof the following (1) to (4):

[0024] (1) a buffer comprising a phosphate buffer at pH 1.0 to 12.0 at aconcentration of 1 mM to 0.5 M;

[0025] (2) a buffer comprising a borate buffer at pH 5.0 to 11.0 at aconcentration of 1 mM to 0.5 M;

[0026] (3) a buffer comprising a Tris-borate buffer at pH 5.0 to 11.0 ata concentration of 1 mM to 0.5 M; and

[0027] (4) a buffer further comprising 0.001 to 1.0% by weight of methylcellulose in the buffer of the above item (3), and wherein the runningbuffer comprises a seaweed extract at a concentration of 0.000001 to0.1% by weight;

[0028] [9] a method of electrophoresis, characterized in that a samplecomprising macromolecular compounds is run in capillary electrophoresisor microchip electrophoresis in the presence of the running buffer ofany one of items [5] to [8] above;

[0029] [10] the method of electrophoresis described in item [9] above,wherein the macromolecular compounds are one kind selected from thegroup consisting of a protein, a peptide, an amino acid, a sugar chain,a polysaccharide and a nucleic acid;

[0030] [11] a method of electrophoresis, characterized by comprising thesteps of injecting a sample comprising macromolecular compounds to acapillary, then applying pressure thereto, and then running the samplein an electric field for electrophoresis such that the macromolecularcompound can be separated, in capillary electrophoresis;

[0031] [12] the method of electrophoresis described in item [11] above,wherein the macromolecular compounds are one kind selected from thegroup consisting of a protein, a peptide, an amino acid, a sugar chain,a polysaccharide and a nucleic acid;

[0032] [13] the method of electrophoresis described in item [11] or [12]above, comprising the steps:

[0033] (a) injecting a sample into a sample injection port of acapillary by application of pressure or an electrical injection, whereinthe capillary comprises the sample injection port and an outlet, and thecapillary is filled with a running buffer [hereinafter referred to asstep (a)]; and

[0034] (b) applying pressure and then running the sample [hereinafterreferred to as step (b)] in capillary electrophoresis;

[0035] [14] the method of electrophoresis described in item [13],wherein the sample is injected into the capillary under the conditionsof no water or no running buffer set in the outlet of the capillary inthe step (a), and wherein pressure is applied to water or buffer in thestep (b):

[0036] [15] the method of electrophoresis described in item [13] or[14], wherein the sample is injected into the capillary by an electricalinjection at 1 to 30 kV for 1 to 30 seconds in the step (a), and whereinthe sample is run in an electric field for electrophoresis of 20 V/cm to10 kV/cm in the step (b);

[0037] [16] the method of electrophoresis described in item [13] or[14], wherein the sample is injected into the capillary by an electricalinjection at 1 to 30 kV for 1 to 60 seconds in the step (a), and whereina pressure of 2 to 50 mbar is applied for 2 to 30 seconds in the step(b);

[0038] [17] the method of electrophoresis according to any one of items[11] or [16] above, wherein the sample is run in the presence of therunning buffer of any one of items [5] to [8] above;

[0039] [18] a method of electrophoresis, characterized by comprising thesteps:

[0040] (A) using a microchip comprising a loading channel, a separationchannel crossing the loading channel, a sample reservoir arranged at oneend of the loading channel, and an outlet arranged at the other end ofthe loading channel, wherein the loading channel and the separationchannel are filled with a running buffer,

[0041] applying voltage or pressure to the loading channel, to supply asample comprising macromolecular compounds from the sample reservoir,thereby introducing the sample into the separation channel [hereinafterreferred to as step (A)]; and

[0042] (B) applying pressure to the separation channel, and then runningthe sample [hereinafter referred to as step (B)];

[0043] [19] the method of electrophoresis described in item [18],wherein the macromolecular compounds are one kind selected from thegroup consisting of a protein, a peptide, an amino acid, a sugar chain,a polysaccharide and a nucleic acid;

[0044] [20] the method of electrophoresis described in item [18] or [19]above, wherein resolution is adjusted by controlling a degree ofpressure applied in the step (B);

[0045] [21] the method of electrophoresis according to any one of items[18] to [20] above, wherein the sample is introduced into the separationchannel by applying voltage to the loading channel under the conditionsof no running buffer set in the outlet in the step (A); and whereinvoltage is applied to the loading channel and the separation channel,thereby running the sample, in the step (B);

[0046] [22] the method of electrophoresis described in item [21] above,wherein a voltage of 10 to 500 V (loading voltage) is applied to theloading channel for 2 to 60 seconds in the step (A); and wherein avoltage of 10 to 500 V (squeezing voltage) is applied to the loadingchannel and, an electric field of 20 V/cm to 50 kV/cm is applied to theseparation channel in the step (B);

[0047] [23] the method of electrophoresis according to any one of items[18] to [20] above, wherein the sample is introduced into the separationchannel by applying pressure to the sample reservoir under theconditions of no running buffer set in the outlet in the step (A); andwherein pressure is applied to the separation channel and then thesample is run in step the (B);

[0048] [24] the method of electrophoresis described in item [23],wherein a pressure of 1 to 1520 mbar is applied to the sample reservoirin the step (A); and wherein a pressure of 1 to 1520 mbar is applied tothe separation channel, and then an electric field of 20 V/cm to 50kV/cm is applied thereto, in step the (B);

[0049] [25] the method of electrophoresis according to any one of items[18] to [24] above, wherein proteins having molecular weights of 9 to205 kDa are separated within 15 seconds;

[0050] [26] the method of electrophoresis according to any one of items[18] to [24] above, wherein sugars comprising 2 to 100 monosaccharidesas a constitutive sacccharide are separated within 15 seconds;

[0051] [27] the method of electrophoresis according to any one of items[18] to [24], wherein nucleic acids of 10 bases to 10 kilobases areseparated within 50 seconds;

[0052] [28] a method for analyzing macromolecular compounds,characterized by comprising the steps of running a sample comprisingmacromolecular compounds by the method of electrophoresis of any one ofitems [9] to [27], thereby separating the macromolecular compounds; andmeasuring mobility by detecting the separated macromolecular compounds;

[0053] [29] the method for analyzing macromolecular compounds describedin item [28] above, wherein the macromolecular compounds are one kindselected from the group consisting of a protein, a peptide, an aminoacid, a sugar chain, a polysaccharide and a nucleic acid; and

[0054] [30] the method for analyzing macromolecular compounds describedin item [28] or [29] above, wherein the separated macromolecularcompounds are detected by at least one kind selected from the groupconsisting of a determination of UV wavelength light absorption, afluorescence detection, an electrochemical detection and achemiluminescence detection.

BRIEF DESCRIPTION OF THE DRAWINGS

[0055]FIG. 1 shows the results obtained when optimal conditions forsample injection and optimal conditions for running voltage for theelectrophoresis were studied in capillary electrophoresis. In theFigure, the individual panels are as follows: panel A, at 5 kV for 5seconds during sample injection, running voltage 3.75 kV; panel B, at 5kV for 5 seconds during sample injection, running voltage 6.5 kV; panelC, at 5 kV for 5 seconds during sample injection, running voltage 15 kV;panel D, at 8 kV for 8 seconds during sample injection, running voltage10 kV; panel E, at 8 kV for 8 seconds during sample injection, runningvoltage 15 kV. Also, the following peaks appeared: 1, Bardykinin (MW:1,060); 2, Angiotensin II (MW: 1,046); 3, α-Melanocyte stimulatinghormone (MW: 1,665); 4, Thyrotropin releasing hormone (MW: 362); 5,Luteinizing hormone releasing hormone (MW: 1,182); 6, Leucine enkephalin(MW: 392); 7, Bombesin (MW: 1,620); 8, Methionine enkephalin (MW: 574);and 9, Oxytocin (MW: 1,007).

[0056]FIG. 2 shows the results of a comparison of the effects of acapillary of 24 cm in effective length and those of a capillary of 8.5cm in effective length on migration time and resolution.

[0057]FIG. 3 shows the results of an examination of the effects ofapplication of pressure on migration time after sample injection andprior to electrophoresis. The respective conditions for applyingpressure are as follows: panel A, no application of pressure aftersample injection; panel B, application of pressure after sampleinjection with the buffer set in the outlet; panel C, and application ofpressure after sample injection, without the buffer set in the outlet.

[0058]FIG. 4 shows the results of a study on sample injectionconditions. The sample was injected without setting the buffer in theoutlet. The respective sample injection conditions are as follows: panelA, no application of pressure after sample injection; panel B,application of pressure at 10 mbar for 6 seconds after sample injection,sample injection port: water, outlet: no buffer; panel C, application ofpressure at 10 mbar for 6 seconds after sample injection, sampleinjection port: water, outlet: water; panel D, application of pressureat 10 mbar for 8 seconds after sample injection, sample injection port:water, outlet: buffer; panel E, and application of pressure at 10 mbarfor 7 seconds after sample injection, sample injection port: buffer,outlet: buffer.

[0059]FIG. 5 shows the results of a study on the effects of time forapplying pressure on migration time and resolution under the conditionsin panel D in FIG. 4. The respective times for applying pressure are asfollows: panel A, at 10 mbar for 2 seconds; panel B, at 10 mbar for 6seconds; and panel C, at 10 mbar for 8 seconds.

[0060]FIG. 6 shows the results of an examination of the effects ofβ-1,3-glucan (curdlan) on migration time and resolution using a systemcontaining β-1,3-glucan (curdlan) as a separation carrier (finalconcentration 0.0001% by weight). Panel A shows the results obtainedunder the conditions without addition of curdlan, and Panel A′ shows theresults under the conditions with addition of curdlan.

[0061]FIG. 7 shows the effects of the addition of methyl cellulose onmigration time and resolution of peptide. Panel A: without addition ofmethyl cellulose, Panel B: with addition of 0.5% methyl cellulose, andPanel C: with addition of 0.1% methyl cellulose.

[0062]FIG. 8 shows the effects of the addition of polysaccharide onmigration time and resolution of peptide. Panel A: without addition ofpolysaccharide, Panel B: with addition of 0.001% curdlan, Panel C: withaddition of 0.0001% curdlan, and Panel D: with addition of 0.0001%seaweed extract.

[0063]FIG. 9 shows the effects of the addition of dextran on migrationtime and resolution of peptide. Panel A: without addition of dextran,Panel B: with addition of 0.5% dextran, Panel C: with addition of 1%dextran, Panel D: with addition of 5% dextran, Panel E: with addition of10% dextran, and Panel F: with addition of 5% dextran. The dextran inpanels A through E had an average molecular weight of approximately100,000 to 200,000, and the dextran in panel F had an average molecularweight of approximately 60,000 to 90,000.

[0064]FIG. 10 shows the effects of the addition of SDS on migration timeand resolution of peptide. Panel A: without addition of SDS, Panel B:with addition of 0.01% SDS, Panel C: with addition of 0.05% SDS, PanelD: with addition of, 0.1% SDS Panel E: with addition of 1% SDS ,andPanel F: with addition of 5% SDS.

[0065]FIG. 11 shows an example of a microchip used for microchipelectrophoresis. 1: sample reservoir, 2: loading channel, 3: outlet, 4:separation channel, and 5: microchip substrate.

[0066]FIG. 12 shows the results of a study on loading conditions andsqueezing conditions in microchip electrophoresis. Panel A shows theresults obtained when a low loading voltage (100 to 300 V), for 10 to 20seconds, and a low squeezing voltage (100 to 400 V) were applied inconventional microchip electrophoresis. Panel B shows the resultsobtained when a high loading voltage (500 V), for 10 to 20 seconds, anda high squeezing voltage (500 V) were applied. Panel C shows the resultsobtained when a loading voltage of 100 V and a squeezing voltage of 100V were applied and a pressure of 50 mbar was applied to the separationchannel prior to electrophoresis, according to the aforementioned VPmethod. Panel D shows the results obtained when the sample was injectedto the loading channel with application of pressure at 150 mbar andthereafter a pressure of 100 mbar was applied to the separation channeland a squeezing voltage (100 V) was applied, according to theaforementioned PP method. Panel E shows the results obtained when thesample was injected to the loading channel with application of pressureat 150 mbar and thereafter a pressure of 150 mbar was applied to theseparation channel and a squeezing voltage (100 V) was applied,according to the aforementioned PP method. In all these cases, theelectric field for electrophoresis was 267 V/cm.

[0067]FIG. 13 shows an electrophoretic pattern obtained by capillaryelectrophoresis using an ordinary capillary.

[0068]FIG. 14 shows the results obtained by subjecting two kinds ofproteins to microchip electrophoresis according to the VP method. PanelA shows the results obtained when bovine insulin (MW: 5733.5) alone waselectrophoresed. Panel B shows the results obtained when myogrobin (MW:16950.9) alone was electrophoresed. Panel C shows the results obtainedwhen both bovine insulin (MW: 5733.5) and myogrobin (MW: 16950.9) wereelectrophoresed.

[0069]FIG. 15 shows the results of a study on the effects of pressure ina step of applying pressure prior to microchip electrophoresis in the VPmethod. Panel A shows the results obtained without application ofpressure as in conventional microchip electrophoresis. Panel B shows theresults obtained with application of low pressure (50 mbar). Panel Cshows the results obtained with application of high pressure (150 mbar).

[0070]FIG. 16 shows the results obtained when bovine insulin and myosinwere subjected to microchip electrophoresis by the VP method. Panels Aand B show the results obtained when bovine insulin and myosin,respectively, were separated by conventional microchip electrophoresis.Panel C shows the results obtained when bovine insulin and myosin wereseparated by the VP method.

[0071]FIG. 17 shows the results of a study on conditions in thedetection of polysaccharides [α-D(+)-galacturonic acid monohydrate,β-1,3-glucan, D-glucronic acid and seaweed extract] for each of the VPmethod and PP method. Panel A shows the results obtained when a voltageof 500 V (loading voltage) was applied for 20 seconds in conventionalmicrochip electrophoresis. Panel B shows the results obtained when avoltage of 300 V (loading voltage) was applied for 10 seconds inconventional microchip electrophoresis. Panel C shows the resultsobtained when an electric field of 500 V (loading voltage) was appliedto the loading channel, to inject the sample and thereafter a lowpressure (50 mbar) was applied to the separation channel, under theconditions of no running buffer in the outlet, in the VP method. Panel Dshows the results obtained when a voltage of 300 V (loading voltage) wasapplied to the loading channel, to inject the sample, and thereafter alow pressure (50 mbar) was applied to the separation channel, under theconditions of no running buffer in the outlet, in the VP method. Panel Eshows the results obtained when a voltage of 300 V (loading voltage) wasapplied to the loading channel, to inject the sample and thereafter amoderate pressure (100 mbar) was applied to the separation channel,under the conditions of no running buffer in the outlet, in the VPmethod. Panel F shows the results obtained when a voltage of 300 V(loading voltage) was applied to the loading channel, to inject thesample and thereafter a high pressure (150 mbar) was applied to theseparation channel, under the conditions of no running buffer in theoutlet, in the VP method. Panel G shows the results obtained when apressure (150 mbar) was applied to the loading channel, to inject thesample and thereafter a low pressure (50 mbar) was applied to theseparation channel, under the conditions of no running buffer in theoutlet, in the PP method. Panel H shows the results obtained when apressure (150 mbar) was applied to the loading channel, to inject thesample and thereafter a high pressure (150 mbar) was applied to theseparation channel, under the conditions of no running buffer in theoutlet, in the PP method.

BEST MODE FOR CARRYING OUT THE INVENTION

[0072] According to the separation carrier of the present invention, itis possible to simplify the procedures, to analyze macromolecularcompounds etc. at high speeds, and to obtain high resolution, incapillary electrophoresis and microchip electrophoresis.

[0073] The term macromolecular compound, as used herein, includesproteins, peptides, amino acids, sugar chains, polysaccharides, nucleicacids (e.g., DNA, RNA, etc.), etc.

[0074] The nucleic acids may be single-stranded or double-stranded.

[0075] The separation carrier of the present invention is a separationcarrier for use in capillary electrophoresis or microchipelectrophoresis, and one of significant features resides in that theseparation carrier comprises at least one kind of compound selected fromthe group consisting of β-glucan and methyl cellulose.

[0076] Because the separation carrier of the present invention containsβ-glucan or methyl cellulose, there are exhibited excellent effects suchthat the adhesion during electrophoresis of proteins, peptides, aminoacids, sugar chains, polysaccharides, nucleic acids (DNA, RNA, etc.),etc. to the wall surface of the capillary used for capillaryelectrophoresis or the wall surface of the channel on the microchip usedfor microchip electrophoresis can be suppressed.

[0077] The aforementioned β-glucan may be a polysaccharide containingβ-1,6 bonds or β-1,4 bonds in the side chain or main chain thereof, aslong as it consists of D-glucose comprising β-glucoside bonds. Theseparation carrier of the present invention includes a compound ormixture containing β-1,3-glucan as a β-glucan, for example, laminaran,curdlan, plant extract, seaweed extract, yeast extract, fungal extractor culture, etc.

[0078] The β-glucan may be used alone or in combination of two or morekinds.

[0079] It is desirable, from the viewpoint of achieving high resolutionand high-speed separation, that the aforementioned β-glucan have anaverage degree of polymerization of 2 or more, preferably 10 or more,and more preferably 20 or more. In addition, from the viewpoint ofobtaining solubility, viscosity and adhesion suitable for separation, itits desirable that the β-glucan has an average degree of polymerizationof 10000 or less, preferably 1000 or less, and more preferably 40 orless.

[0080] The average degree of polymerization as used herein refers to avalue obtained by calculating the polymer/monomer molecular weightratio.

[0081] β-glucan mixtures include, for example, seaweed extract; extractfrom mushroom such as Agaricus, Lentinula edodes, Pleurotus osteatus,Hericium erinareum, Sparassis crispa, Shizophyllum commune Fries andCoriolus versicolor; extract from plants such as barley, wild oat andoat; and extract from microorganisms such as baker's yeast, brewer'syeast, mycobacteria, fungi, filamentous fungi, Chlorella and microalgaeor cultures thereof.

[0082] The aforementioned seaweed extract is obtained from a seaweed(Undaria, Laminariales, Eisenia, etc.) by, for example, a methoddescribed in the literature (Japanese Patent Application No. 2000-229369etc.). Specifically, the aforementioned seaweed extract can be obtained,for example, by subjecting a seaweed (Undaria, Laminariales, Eisenia,etc.) to water extraction (extraction with hot water, warm water, coldwater, ice-cooled water, etc.), acid/alkali extraction at varioustemperatures, dissolution in a solvent (ethanol, methanol, acetone,ether, etc.), etc.

[0083] The aforementioned seaweed extract is a mixture of, for example,water-soluble laminaran, which is a β-1,3-glucan having a large numberof β-1,6 bonds, or slightly soluble laminaran, which is a β-1,3-glucanhaving a small number of β-1,6 bonds.

[0084] It is desirable, from the viewpoint of obtaining solubility,viscosity, and adhesion suitable for separation, that the aforementionedmethyl cellulose have an average degree of polymerization of 10 or more,preferably 180 or more, and more preferably 1800 or more. In addition,it is desirable from the viewpoint of obtaining sufficient resolutionthat the methyl cellulose has an average degree of polymerization of30000 or less, preferably 10000 or less, and more preferably 3000 orless.

[0085] The aforementioned methyl cellulose may be a derivative thereof.The derivatives include, for example, hydroxymethyl cellulose,hydroxymethylpropyl cellulose, hydroxypropyl cellulose, hydroxyethylcellulose, carboxylmethyl cellulose, etc.

[0086] The separation carrier of the present invention makes it possibleto simplify the procedures, to analyze macromolecular compounds,specifically, proteins, peptides, sugar chains, polysaccharides, etc.,at high speeds, and obtain high resolution, in capillary electrophoresisand microchip electrophoresis, when added to the running buffer inelectrophoresis.

[0087] Accordingly, the present invention provides a running buffercontaining the aforementioned separation carrier.

[0088] The running buffer of the present invention is a running bufferused for capillary electrophoresis or microchip electrophoresis, and oneof features resides in that the aforementioned separation carrier iscontained therein.

[0089] According to such a running buffer, it is possible to suppressthe adhesion during electrophoresis of analytes such as proteins,peptides, amino acids, sugar chains, polysaccharides or nucleic acids tothe wall surface of the capillary used for capillary electrophoresis orthe wall surface of the channel on the microchip used for microchipelectrophoresis. Hence, the running buffer of the present inventionenables capillary electrophoresis and microchip electrophoresis to besuitable for the migration of macromolecular compounds, to be simple inoperation, and to obtain high resolution at high speeds.

[0090] The term “analyte,” as used herein, refers to a substance to beanalyzed contained in the sample, i.e., a macromolecular compound. Theterm simply referred to as “sample” means a mixture containing themacromolecular compound, etc.

[0091] It is desirable, from the viewpoint of obtaining high resolutionat high speeds, that the running buffer of the present inventioncontains a phosphate buffer at pH 1.0 to 12.0, preferably pH 2.0 to 4.0,at a concentration of 1 mM to 0.5 M, preferably 10 mM to 0.5 M, when theanalyte is a peptide.

[0092] From the viewpoint of sufficiently exhibiting an effect ofresolution, the concentration of the phosphate buffer in the runningbuffer of the present invention is preferably 1 mM or more, morepreferably 10 mM or more, still more preferably 50 mM or more, and yetmore preferably 75 mM or more. In addition, from the viewpoint ofexhibiting an effect of shortening migration time and obtaining highresolution, the concentration of the phosphate buffer in the runningbuffer of the present invention is preferably 0.5 M or less, morepreferably 0.2 M or less, and still more preferably 0.1 M or less.

[0093] It is preferable, from the viewpoint of buffer capacity andresolution in electrophoresis, that the pH of the aforementionedphosphate buffer is preferably 1.0 to 12.0, more preferably 2.0 to 4.0,still more preferably 2.0 to 2.8, yet more preferably 2.5 to 2.8, andespecially preferably 2.5.

[0094] When the analyte is, for example, a protein, it is desirable thatthe running buffer of the present invention contains a borate buffer atpH 5.0 to 11.0, preferably pH 7.0 to 9.6, at a concentration of 1 mM to0.5 M, preferably 10 mM to 0.15 M. Here, it is preferable, from theviewpoint of sufficiently exhibiting an effect of resolution, that theconcentration of the borate buffer in the running buffer of the presentinvention is 1 mM or more, more preferably 2.0 mM or more, still morepreferably 10 mM or more, yet more preferably 20 mM or more, andespecially preferably 40 mM or more, and from the viewpoint ofexhibiting an effect of shortening migration time and obtaining highresolution, the concentration of the borate buffer in the running bufferof the present invention is preferably 0.5 M or less, more preferably0.15 M or less, still more preferably 0.1 M or less, and yet morepreferably 75 mM or less. It is preferable, from the viewpoint of buffercapacity and resolution in electrophoresis, that the pH of theaforementioned borate buffer is 5.0 to 11.0, more preferably 7.0 to 9.5,and still more preferably 8.0 to 9.0.

[0095] When the analyte is, for example, a sugar or polysaccharide, itis desirable that the running buffer of the present invention contains aTris-borate buffer at pH 5.0 to 11.0, preferably pH 7.0 to 9.6, at aconcentration of 1 mM to 0.5 M, preferably 10 mM to 0.5 M, and morepreferably 10 mM to 0.15 M. Here, it is preferable, from the viewpointof sufficiently exhibiting an effect of resolution, that theconcentration of the Tris-borate buffer in the running buffer of thepresent invention is 1 mM or more, more preferably 10 mM or more, stillmore preferably 20 mM or more, and yet more preferably 40 mM or more,and from the viewpoint of exhibiting an effect of shortening migrationtime and obtaining high resolution, the concentration of the Tris-boratebuffer in the running buffer of the present invention is preferably 0.5M or less, more preferably 0.25 M or less, still more preferably 0.15 Mor less, yet more preferably 0.1 M or less, and especially preferably 75mM or less. Also, it is preferable, from the viewpoint of buffercapacity and resolution in electrophoresis, that the pH of theaforementioned Tris-borate buffer is 5.0 to 11.0, more preferably 7.0 to9.5, still more preferably 8.0 to 9.0, and yet more preferably 8.0 to9.0.

[0096] When the analyte is, for example, a nucleic acid, it is desirablethat the above buffer comprising Tris-borate buffer at 10 mM to 0.5 M,preferably 10 mM to 0.15 M, comprises methyl cellulose at 0.001 to 0.7%by weight, preferably 0.05 to 0.7% by weight.

[0097] The concentration of the separation carrier in the running buffercomprising the separation carrier can be appropriately set according tothe kind of compound used. In the case of methyl cellulose, for example,it is preferable, from the viewpoint of improvement of resolution, thatthe concentration of the separation carrier is 0.001 to 1.0% by weight,more preferably 0.7% by weight, still more preferably 0.05 to 0.5% byweight, and especially preferably 0.1% by weight.

[0098] When the separation carrier is curdlan, it is preferable, fromthe viewpoint of high-speed separation, improvement of resolution, andprevention of analyte adsorption to the wall surface, that itsconcentration is 0.000001 to 0.1% by weight, more preferably 0.00001 to0.01% by weight, and still more preferably 0.0001 to 0.001% by weight.

[0099] When the separation carrier is seaweed extract, it is preferable,from the viewpoint of high-speed separation, improvement of resolution,and prevention of analyte adsorption to the wall surface, that itsconcentration is 0.000001 to 0.1% by weight, more preferably 0.00001 to0.001% by weight, and especially preferably 0.0001% by weight.

[0100] According to the running buffer of the present invention, thereis provided a method of electrophoresis that is based on capillaryelectrophoresis or microchip electrophoresis, that is suitable for theelectrophoresis of macromolecular compounds, that is simple inoperation, and that offers high resolution at high speeds.

[0101] The methods of electrophoresis of the present invention include,particularly the followings:

[0102] (1) a method characterized by electrophoresing a samplecomprising a macromolecular compound in the presence of theaforementioned running buffer in capillary electrophoresis or microchipelectrophoresis;

[0103] (2) a method of electrophoresis characterized by injecting asample comprising a macromolecular compound to the capillary andthereafter applying pressure and electrophoresing the sample in anelectric field for electrophoresis enabling the separation of themacromolecular compound, in capillary electrophoresis; and

[0104] (3) a method characterized by performing in microchipelectrophoresis a process comprising the following steps:

[0105] (A) using a microchip comprising a loading channel, a separationchannel crossing the loading channel, a sample reservoir arranged at oneend of the loading channel, and an outlet arranged at the other end ofthe loading channel, wherein the loading channel and the separationchannel are filled with a running buffer,

[0106] applying voltage or pressure to the loading channel, to supply asample comprising macromolecular compounds from the sample reservoir,thereby introducing the sample into the separation channel [hereinafterreferred to as step (A)]; and

[0107] (B) applying pressure to the separation channel, and then runningthe sample [hereinafter referred to as step (B)].

[0108] A sample applicable to methods of electrophoresis of the presentinvention includes samples containing macromolecular compounds,specifically a sample comprising a protein, a peptide, an amino acid, asugar chain, a polysaccharide, a nucleic acid (e.g., DNA, RNA, etc.), orthe like. Such a “sample” includes, but is not limited to, a sample fromorganism.

[0109] In the case of capillary electrophoresis, it is desirable, fromthe viewpoint obtaining good resolution and shortening migration time,that the electric field for electrophoresis is 20 V/cm to 10 kV/cm,preferably 50 V/cm to 5 kV/cm, and more preferably 100 V/cm to 1 kV/cm.

[0110] Also, in the case of microchip electrophoresis, it is desirable,from the viewpoint of obtaining good resolution and shortening migrationtime, that the electric field for electrophoresis is 20 V/cm to 50kV/cm, preferably 50 V/cm to 20 kV/cm, and more preferably 100 V/cm to10 kV/cm.

[0111] With regard to the capillary used for the aforementionedcapillary electrophoresis, inner diameter, outer diameter, total lengthand effective length are not particularly limited; for effective length,in particular, a capillary of short effective length can be used toenable analysis at high speeds. Here, the effective length of acapillary refers to the distance from the sample injection port to thedetection portion.

[0112] In the aforementioned microchip electrophoresis, there can beused a microchip comprising a loading channel, and a separation channelcrossing the loading channel, a sample reservoir arranged at one end ofthe loading channel and an outlet arranged at the other end of theloading channel. An example of such a microchip is shown in FIG. 11. InFIG. 11, the loading channel 2 and the separation channel 4 cross toeach other on the microchip substrate 5, with the sample reservoir 1arranged at one end of the loading channel 2 and the outlet 3 arrangedat the other end.

[0113] A material for the aforementioned microchip substrate includes,for example, quartz glass, borosilicate glass, soda glass, polymethylmethacrylate, polycarbonate, dimethyl siloxane, etc. Among them, glassand polymethacrylate are desirable from the viewpoints of low adsorptionof sample and easy processing of chip.

[0114] The size of the aforementioned microchip is, for example, 10 to120 mm in length, 10 to 120 mm in width, and 500 to 5000 μm inthickness.

[0115] The shapes of the loading channel and separation channel in theaforementioned microchip are not particularly limited.

[0116] The width of the aforementioned channel can be appropriately setaccording to the size, purpose of use, etc. of the microchip.Specifically, it is desirable, from the viewpoint of obtainingsufficient analytical sensitivity, that the width of the aforementionedchannel is 0.1 μm or more, preferably 10 μm or more. In addition, it isdesirable, from the viewpoint of sufficient analytical accuracy, thatthe width of the aforementioned channel is 100 μm or less, preferably 50μm or less. Also, the depth of the aforementioned channel can beappropriately set according to the size, purpose of use, etc. of themicrochip; it is desirable, from the viewpoint of obtaining sufficientanalytical sensitivity, that the depth of the aforementioned channel is0.1 μm or more, preferably 10 μm or more. In addition, it is desirable,from the viewpoint of sufficient analytical accuracy, that the width ofthe aforementioned channel is 100 μm or less, preferably 50 μm or less.Further, although the length of the aforementioned separation channelcan be appropriately set according to the size of the microchip and thecompound to be analyzed, it is desirable that the effective length islonger. Effective length refers to the distance from the part in whichchannels are crossed, to the macromolecular compound detection point(arranged on the separation channel). It is desirable, from theviewpoint of obtaining sufficient resolution, that the effective lengthis 0.1 mm or more, preferably 10 mm or more. In addition, it isdesirable, from the viewpoint of high-speed separation, that theeffective length is 100 mm or less, preferably 50 mm or less.

[0117] Also, the size of the aforementioned reservoir can beappropriately set according to the sample volume. Specifically, it isdesirable, from the viewpoints of handling during sample introductionand electrode thickness, that the diameter of the reservoir is 0.05 mmor more, preferably 1 mm or more, and it is desirable from the viewpointof the amount of sample used that the diameter is 5 mm or less,preferably 3 mm or less.

[0118] In microchip electrophoresis, it is desirable, from the viewpointof obtaining good resolution, that the amount (concentration) of sampleinjected is 0.1 ng/ml to 1 g/ml, preferably 10 ng/ml to 100 mg/ml, andmore preferably 0.1 μg/ml to 10 mg/ml, when the sample is a peptide orprotein. When the aforementioned sample is a sugar or polysaccharide, itis desirable, from the viewpoint of obtaining good resolution, that theamount (concentration) of sample injected is 0.1 μg/ml to 10 g/ml,preferably 1 mg/ml to 5 g/ml, and more preferably 100 mg/ml to 1 g/ml.When the aforementioned sample is a nucleic acid, it is desirable, fromthe viewpoint of obtaining good resolution, that the amount(concentration) of sample injected is 1 ng/ml to 500 μg/ml, preferably10 ng/ml to 100 μg/ml, and more preferably 100 ng/ml to 50 μg/ml.

[0119] In the method of electrophoresis (1) above, running voltage etc.during electrophoresis can be appropriately set according to thecompound to be analyzed [protein, peptide, amino acid, sugar chain,polysaccharide, nucleic acid (e.g., DNA, RNA, etc.), etc.]. Such runningvoltage can be determined according to, for example, resolution of asample, the viscosity of the running buffer used, the number of analytescontained in the sample, etc.

[0120] The method of electrophoresis (2) above includes, particularly amethod comprising performing in capillary electrophoresis a processcomprising: (a) injecting a sample into a sample injection port of acapillary by application of pressure or an electrical injection, whereinthe capillary comprises the sample injection port and an outlet, and thecapillary is filled with a running buffer, while a voltage or a pressureapplied [hereinafter referred to as step (a)], and (b) applying pressureby using water or a buffer, and thereafter running the sample[hereinafter referred to as step (b)].

[0121] Here, the running buffer includes 10 mM to 0.5 M phosphate bufferat pH 1.0 to 12.0, preferably pH 2.0 to 4.0; 10 mM to 0.5 M, preferably10 mM to 0.15 M borate buffer at pH 5.0 to 11.0, preferably pH 7.0 to9.5; 10 mM to 0.5 M, preferably 10 mM to 0.15 M Tris-borate buffer at pH5.0 to 11.0, preferably pH 7.0 to 9.5; running buffers containing theseparation carrier of the present invention, etc. From the viewpoint ofobtaining high-speed analysis and high resolution, running bufferscontaining the separation carrier of the present invention arepreferred.

[0122] In step (a) above, it is preferable, from the viewpoint ofobtaining high-speed analysis and high resolution, that the sample isinjected under the conditions of no running buffer set in the outlet ofthe capillary.

[0123] A means of electric injection in step (a) above includes applyingan electric field suitable for sample injection in the capillary.

[0124] In step (b) above, as a means of electric injection, there may bementioned applying a pressure suitable for shortening apparent effectivelength, wherein electrophoresis is performed under an running voltagesuitable for the separation of macromolecular compounds in the sample.Running voltage in step (b) above can be determined according to thecompound to be analyzed [protein, peptide, amino acid, sugar chain,polysaccharide, nucleic acid (e.g., DNA, RNA, etc.), etc.], resolutionof a sample, the viscosity of the running buffer used, and the number ofanalytes contained in the sample.

[0125] The method of electrophoresis (2) above can specifically beconducted by, for example, injecting the sample to the capillary at avoltage of 1 to 30 kV, preferably 5 to 15 kV, for 1 to 30 seconds,preferably 5 to 15 seconds, in step (a), and

[0126] applying pressure to water or a buffer at 2 to 50 mbar for 2 to30 seconds and separating the analyte under an electrophoretic field of20 V/cm to 50 kV/cm, in step (b).

[0127] Also, sample injection conditions and conditions for applyingpressure can be appropriately set according to the kind and performanceof apparatus, the shape of injection portion, the shape and size ofsample vial, the material and shape of sample cap, sample viscosity andconcentration, etc.

[0128] As the method of electrophoresis (3) above, there mayspecifically be mentioned a method comprising performing a processcomprising applying a voltage to the loading channel under theconditions of no running buffer set in the outlet in step (A), andapplying pressure to the separation channel, and then applying pressureto the loading channel and applying a voltage to the separation channel,thereby running the sample in step (B) [hereinafter referred to asprocess 1]; or a process comprising applying pressure to the samplereservoir, to introduce the sample comprising macromolecular compoundsto the separation channel under the conditions of no running buffer setin the outlet in the step (A), and applying pressure to the separationchannel, and then running the sample in the step (B) [hereinafterreferred to as process 2].

[0129] According to method (3) above, there are exhibited excellenteffects such that proteins having molecular weights of 9 to 205 kDa canbe separated within 15 seconds, sugars having 2 to 100 constituentmonosaccharides can be separated within 15 seconds, and DNA of 10 baseto 10 kilobase can be separated within 50 seconds, because the methodcomprises the aforementioned steps.

[0130] In process 1 above, a voltage (or an electric field) suitable forthe viscosities of the sample introduced and buffer used is applied inthe step (A).

[0131] Specifically, a voltage of 100 to 500 V is applied to the loadingchannel in the step (A), and a pressure of 1 to 1520 mbar, preferably 10to 760 mbar, is applied to the separation channel, and thereafter avoltage of 100 to 500 V is applied to the loading channel and anelectric field of 20 V/cm to 50 kV/cm is applied to the separationchannel in the step (B).

[0132] Resolution can be adjusted by regulating the pressure duringapplication of pressure in the step (B).

[0133] In process 2, application of pressure in the step (A) includespressure suitable for the viscosities of the sample to be introduced andthe buffer.

[0134] Specifically, a pressure of 1 to 1520 mbar, preferably 50 to 760mbar is applied to the sample reservoir in the step (A), and a pressureof 1 to 1520 mbar, preferably 10 to 760 mbar is applied to theseparation channel and thereafter an electric field of 20 V/cm to 50kV/cm is applied in the step (B).

[0135] Resolution can be adjusted by regulating the pressure duringapplication of pressure in the step (B).

[0136] Therefore, it is desirable that the apparatus used for the methodof electrophoresis of the present invention is equipped with anapparatus for applying pressure. The present invention encompasseselectrophoresis apparatuses suitable for use in the method ofelectrophoresis of the present invention. Such apparatuses include, butnot limited to, an apparatus comprising a capillary or microchip; ameans for holding the capillary or microchip; a means for applying anelectric field or a voltage to the capillary or microchip; a means forapplying pressure to the capillary or microchip; a power supply forsupplying an appropriate electricity to these means; a transformer; ameans for regulating the electric field, voltage or pressure (computeretc.); a means for injecting a sample to the capillary or microchip;etc., and the like.

[0137] According to the method of electrophoresis of the presentinvention, there is provided a method for analyzing macromolecularcompounds by which high resolution can be obtained simply and quickly,and which is applicable to proteome analysis, glycome analysis, etc. Agreat feature of such an analytical method resides in that the methodcomprises running a sample comprising macromolecular compounds, toseparate the macromolecular compounds, and then detecting the separatedmacromolecular compounds, to measure their mobility.

[0138] In the analytical method of the present invention, amacromolecular compound in the sample can be detected by, for example,UV wavelength light absorption, fluorescence, laser, lamps, LED, etc.,or by electrochemical measurement or chemiluminescence measurement.Specifically, when the macromolecular compound is a protein or peptide,the protein or peptide can be detected by measuring absorbance at 200nm; by reacting SYPRO Orange and the protein or peptide, exciting at 460to 550 nm, and determining fluorescence at 550 to 650 nm; and byelectrochemical measurement, chemiluminescence measurement, etc. Whenthe macromolecular compound is a sugar chain or polysaccharide, thesugar chain or polysaccharide can be detected by measuring absorbance at260 nm or 280 nm; by reacting SYPRO Orange and the sugar chain orpolysaccharide, and determining fluorescence; and by electrochemicalmeasurement, chemiluminescence measurement, etc.

[0139] In the aforementioned capillary electrophoresis, for example, anapparatus capable of generating UV wavelength light and a detector forthe UV wavelength light may be arranged, or an apparatus capable ofgenerating fluorescence and a detector for the fluorescence may bearranged, in the outlet of the capillary.

[0140] In the aforementioned microchip electrophoresis, for example, adetector for UV wavelength light may be arranged, or an apparatuscapable of generating fluorescence and a detector for the fluorescencemay be arranged, at a detection point on the separation channel.

[0141] In the detection, when a protein, a peptide, an amino acid, asugar chain, a polysaccharide, a nucleic acid (DNA, RNA, etc.), etc.,can be achieved by UV absorption, comparison of migration time withstandards, mass spectrometry, etc.

[0142] The present invention is hereinafter described in more detail bymeans of, but not limited to, the following examples.

PRODUCTION EXAMPLE 1

[0143] According to the method as described in the literature [e.g.,Japanese Patent Application No. 2000-229369 etc.], a seaweed such asUndaria (including sporophyll or stem or root or frond), Laminariales orEisenia was extracted with hot water to yield an extract.

[0144] Subsequently, the extract was dissolved in deionized water. Theresulting solution was subjected to capillary electrophoresis to analyzethe composition of the extract.

[0145] For the capillary electrophoresis, the HP^(3D) CE systemmanufactured by Hewlett Packard Company was used. The capillary used wasthe DB-17 capillary of which inner wall was coated with diphenyldimethyl polysiloxane [inner diameter: 0.1 mm, outer diameter: 0.36 mm,total length: 32.5 cm, effective length: 24 cm; manufactured by J&WScientific]. Detection was carried out at 260 nm and 280 nm using aphotodiode array.

[0146] The conditions for the capillary electrophoresis were capillarytemperature: 25° C., electric field: 200 or 300 V/cm, injectionconditions: at 5 kV for 5 seconds.

[0147] As a result, when the electric field was 300 V/cm, fraction 1showing a maximum absorption at a wavelength of 260 nm was obtained atmigration times of 3.5 to 4 minutes, and fraction 2 showing a maximumabsorption at a wavelength of 280 nm was obtained at migration times of4 to 6 minutes.

[0148] Based on comparisons with standards showed that fraction 1 abovewas water-soluble laminaran mainly composed of a β-1,3-glucan containingβ-1,6 bonds, and fraction 2 above was a polysaccharide containinggalactose or uronic acid.

EXAMPLE 1

[0149] Electrophoretic conditions in capillary electrophoresis werestudied. Peptide standards manufactured by Bio-Rad Laboratories Inc.[Bardykinin (MW: 1,060), Angiotensin II (MW: 1,046), α-Melanocytestimulating hormone (MW: 1,665), Thyrotropin releasing hormone (MW:362), Luteinizing hormone releasing hormone (MW: 1,182), Bombesin (MW:1,629), Leucine enkephalin (MW: 392), Methionine enkephalin (MW: 574),and Oxytocin (MW: 1,007)] were dissolved in deionized water(manufactured by ICN Biomedicals Inc.) to give a final concentration of50 μg/ml. The solutions obtained were used as the peptide samples in thefollowing procedures.

[0150] For the capillary electrophoresis, the HP^(3D) CE systemmanufactured by Hewlett Packard Company was used. The capillary used wasthe DB-17 capillary of which inner wall was coated with diphenyldimethyl polysiloxane [inner diameter: 0.1 mm, outer diameter: 0.36 mm,total length: 32.5 cm, coating layer thickness: 0.1 μm, manufactured byJ&W Scientific]. Peptide detection was carried out with absorption at200 nm as an index. The conditions for the capillary electrophoresiswere method: CZE, electrode setting: anode-cathode, capillarytemperature: 25° C., and running voltage: 10 kV. The running buffer usedwas 0.1 M phosphate buffer (pH 2.5).

[0151] First, conditions for sample injection and running voltageconditions for electrophoresis were studied. This investigation wasconducted at 5 to 8 kV for 5 to 8 seconds for sample injection and at 5to 15 kV for running voltage. FIG. 1 shows the results of an examinationof the effects of sample injection conditions and running voltage onmigration time and separation of peptides in a capillary of 24 cm ineffective length.

[0152] As a result, as shown in panels D and E, it was shown thatsufficient intensity was obtained with sample injection at 8 kV for 8seconds.

[0153] It was suggested that the conditions of injecting the sample at 8kV for 8 seconds and electrophoresing the sample at an running voltageof 10 kV may be the most suitable for obtaining a sharp peak and goodresolution and shortening the migration time.

[0154] Sufficient intensity was also obtained under the conditions ofinjecting the sample at 5 kV for 5 seconds and electrophoresing thesample at an running voltage of 6.5 kV, or by sample injection at 8 kVfor 8 seconds and electrophoresis at an running voltage of 15 kV.

[0155] Based on the above results, effective length of capillary wasstudied using a capillary of 24 cm or 8.5 cm in effective length. FIG. 2shows the results of an examination of the effects of effective lengthof capillary on migration time and separation of peptides.

[0156] As a result, it was shown that migration time could be shortenedwithout affecting the resolution with the use of the 24 cm capillary,even when using the capillary of 8.5 cm in effective length.

EXAMPLE 2

[0157] The migration time-shortening effect of application of pressureafter sample injection was examined. Specifically, sample injection wasconducted under the conditions with or without water or buffer set inthe sample injection port or outlet, and thereafter sample migrationtime and resolution were examined without or with application ofpressure (10 mbar, 8 seconds). The samples used were the peptide samplesin Example 1 above. The results are shown in FIG. 3.

[0158] Panel A shows an electrophoresis pattern obtained by aconventional method (no application of pressure after sample injection).Panel B shows the results obtained under the conditions with applicationof pressure (10 mbar, 8 seconds) after sample injection and withphosphate buffer (pH 2.5) set in the outlet of the capillary. Panel Cshows the results obtained under the conditions with application ofpressure (10 mbar, 8 seconds) after sample injection and withoutphosphate buffer (pH 2.5) set in the outlet of the capillary.

[0159] As a result, it was shown that good resolution could be obtainedand migration time could be shortened when sample injection wasconducted under the conditions without buffer set in the outlet of thecapillary and with application of pressure after sample injection.

[0160] Furthermore, resolution and migration time were examined whensample injection was conducted under the conditions with buffer set inthe outlet of the capillary, and thereafter pressure was applied at 10mbar for 6 to 8 seconds under the conditions with or without water orbuffer set in the sample injection port or outlet. The results are shownin FIG. 4.

[0161] Panel A shows the results obtained under the conventionalconditions wherein pressure is not applied after sample injection.Panels B through D show the results obtained under the conditionswherein sample injection was conducted without buffer set in the outletof the capillary and pressure was applied with water set in the sampleinjection port of the capillary. Panel E shows the results obtainedunder the conditions wherein sample injection was conducted withoutbuffer set in the outlet of the capillary and pressure was applied withbuffer set in the sample injection port of the capillary. The respectiveconditions for applying pressure are shown below.

[0162] Conditions in panel B

[0163] Sample injection followed by application of pressure at 10 mbarfor 6 seconds, sample injection port: water, outlet: no buffer

[0164] Conditions in panel C

[0165] Sample injection followed by application of pressure at 10 mbarfor 6 seconds, sample injection port: water, outlet: water

[0166] Conditions in panel D

[0167] Sample injection followed by application of pressure at 10 mbarfor 8 seconds, sample injection port: water, outlet: buffer

[0168] Conditions in panel E

[0169] Sample injection followed by application of pressure at 10 mbarfor 7 seconds, sample injection port: buffer, outlet: buffer

[0170] As a result, as shown in panel D in FIG. 4, it was shown thatmigration time could be shortened and good resolution could be obtainedunder the same conditions as in panel C in FIG. 3 above.

[0171] Furthermore, the effects of time for applying pressure onmigration time and resolution were examined under the conditions inpanel D in FIG. 4. Some of the results are shown in FIG. 5.

[0172] The conditions for applying pressure were panel A: at 10 mbar for2 seconds; panel B: at 10 mbar for 6 seconds; and panel D: at 10 mbarfor 8 seconds.

[0173] As shown in FIG. 5, it can be seen that sharp peaks are obtainedwith time for applying pressures exceeding 6 seconds (7 to 8 seconds).

EXAMPLE 3

[0174] The effects of β-1,3-glucan (curdlan) on migration time andresolution were examined using a system comprising a running buffer andcurdlan added thereto as a separation carrier (final concentration:0.0001% by weight) under the conditions of conducting sample injectionwithout phosphate buffer (pH 2.5) set in the outlet of a capillary of8.5 cm in effective length, and thereafter applying pressure (10 mbar, 8seconds). The samples used were the peptide samples in Example 1 above.

[0175] As a result, as shown in FIG. 6, it can be seen that sharperpeaks are obtained and separation in shorter times is possible in thepresence of 0.0001% by weight curdlan as a separation carrier than inthe absence thereof.

[0176] Although peptide analysis takes 25 minutes using a capillary of24 cm in effective length, the method using 0.0001% by weight curdlan asa separation carrier makes it possible to analyze peptides in 5 minutesand obtain good resolution using a capillary of 8.5 cm in effectivelength.

EXAMPLE 4

[0177] Running buffers suitable for protein analysis were studied bycapillary electrophoresis.

[0178] Using running buffers at concentrations ranging from 0.5 to 0.1 M[phosphate buffer (pH 2.5), borate buffer (pH 5.5) or Tris-borate buffer(pH 8.5)], the optimal conditions for protein sample separation werestudied.

[0179] For the capillary electrophoresis, the HP^(3D) CE systemmanufactured by Hewlett Packard Company was used. The capillary used wasthe DB-17 capillary of which inner wall was coated with diphenyldimethyl polysiloxane [inner diameter: 0.1 mm, outer diameter: 0.36 mm,total length: 32.5 cm, effective length: 24 cm; manufactured by J&WScientific]. Peptide detection was carried out with absorption at 200 nmas an index. The conditions for the capillary electrophoresis weremethod: CZE, electrode setting: anode-cathode, capillary temperature:25° C., and running voltage: 10 kV.

[0180] The samples used were the peptide samples described in Example 1.

[0181] As a result, when using phosphate buffers (pH 2.5) atconcentrations of 0.1 M or less, shortened migration time and improvedresolution were observed.

EXAMPLE 5

[0182] Optimal conditions for separation of protein and peptide sampleswere studied using as separation carriers methyl cellulose [averagemolecular weight: approximately 400,000, manufactured by Sigma Company],dextran [average molecular weight: approximately 100,000 to 200,000 and60,000 to 90,000, both manufactured by Wako Pure Chemical Industries,Ltd.], curdlan [manufactured by Wako Pure Chemical Industries, Ltd.],sodium dodecyl sulfate (SDS, manufactured by Wako Pure ChemicalIndustries, Ltd.), sodium alginate [manufactured by Nacalai Tesque,Inc.] and the seaweed extract obtained in Production Example 1 above.

[0183] For the capillary electrophoresis, the HP^(3D) CE systemmanufactured by Hewlett Packard Company was used. The capillary used wasthe DB-17 capillary of which inner wall was coated with diphenyldimethyl polysiloxane [inner diameter: 0.1 mm, outer diameter: 0.36 mm,total length: 32.5 cm, effective length: 24 cm, manufactured by J&WScientific]. Peptide detection was carried out with absorption at 200 nmas an index. The conditions for the capillary electrophoresis weremethod: CZE, electrode setting: anode-cathode, capillary temperature:25° C., and running voltage: 10 kV. The running buffer used was 75 mMphosphate buffer (pH 2.5). The samples used were the peptide samplesdescribed in Example 1. The results are shown in FIGS. 7 through 10.

[0184] As a result, it was shown that migration time could be shortenedand good resolution could be obtained by the addition of 0.1% by weightmethyl cellulose (panel C in FIG. 7), by the addition of 0.0001 to0.001% by weight curdlan (panels B and C in FIG. 8), and by the additionof 0.0001% by weight seaweed extract (panel D in FIG. 8).

EXAMPLE 6

[0185] Electrophoretic conditions in microchip electrophoresis werestudied.

[0186] For the microchip electrophoresis, a microchip electrophoresisapparatus [SV1100, manufactured by Hitachi Electronics Co., Ltd.]equipped with an LED detector was used to evaluate separation ofproteins in the microchip. The microchip used was the microchip in thei-chip kit developed for DNA analysis [manufactured by Hitachi ChemicalCo., Ltd.]. This microchip was made from poly(methyl methacrylate)(PMMA), and comprises a loading channel of 50 μm in width, 30 μm indepth and 8 mm in length, a separation channel of 50 μm in width, 30 μmin depth and 30 mm in length, and a reservoir [see FIG. 11].

[0187] Procedures for microchip electrophoresis are described below.

[0188] 1) VP Method

[0189] The aforementioned channel was filled with a buffer and a sample.Subsequently, a voltage of 10 to 500 V (loading voltage) was applied tothe loading channel for 10 to 60 seconds under the conditions of norunning buffer in the outlet [corresponding to 3 in FIG. 11], to injectthe sample. After applying a pressure (1 to 520 mbar) to the separationchannel, electrophoresis was conducted by applying a voltage of 10 to500 V (squeezing voltage) and a voltage of 30 to 900 V (running voltage)(electric field of 100 to 300 V/cm), thereby separating the sample.

[0190] 2) PP Method

[0191] Sample injection was conducted by applying a pressure (1 to 1520mbar), in place of sample injection to the separation channel byapplying a voltage, in the aforementioned VP method. After applying apressure (1 to 1520 mbar) to the separation channel, electrophoresis wasconducted by applying a voltage of 300 to 900 V (running voltage)(electric field of 100 to 300 V/cm), thereby separating the sample.

[0192] 3) Conventional Microchip Electrophoresis

[0193] The aforementioned channel and well were filled with a buffer anda sample. A voltage of 100 to 500 V was applied to the loading channelfor 20 seconds. Subsequently, a voltage of 100 to 500 V was applied tothe crossing portion of the microchip and a voltage of 300 to 900 V(running voltage) (electric field of 100 to 300 V/cm) was applied to theseparation channel, thereby separating the sample.

[0194] (1) Protein Detection

[0195] Lysozyme (MW: 14,400), Trypsin inhibitor (MW: 21,500), Carbonicanhidrorase (MW: 31,000), Ovalbumin (MW: 45,000), Serum albumin (MW:66,200), Phosphorylase B (MW: 97,000), β-Galactosidase (MW: 116,000),and Myosin (MW: 200,000) [all manufactured by Bio-Rad Laboratories Inc.]were used as protein standards. Each of these protein standards wasdissolved in deionized water to obtain a protein solution having a finalconcentration of 0.3 μg to 7.5 mg/ml.

[0196] The running buffer for proteins used was 0.05 M borate buffer (pH9.3).

[0197] In the microchip, 1 μl of SYPRO Orange was reacted with 10 μl ofeach protein solution. Proteins were detected by absorption at 220 nm.

[0198] The results are shown in FIG. 12. In FIG. 12, panel A shows theresults obtained when a low loading voltage (100 to 300 V), for 10 to 20seconds, and a low squeezing voltage (100 to 400 V) were applied inconventional microchip electrophoresis. Panel B shows the resultsobtained when a high loading voltage (500 V), for 10 to 20 seconds, anda high squeezing voltage (500 V) were applied. Panel C shows the resultsobtained when a loading voltage of 100 V and a squeezing voltage of 100V were applied, and a pressure of 50 mbar was applied to the separationchannel prior to electrophoresis, according to the aforementioned VPmethod. Panel D shows the results obtained when a mixture of eight kindsof proteins was analyzed according to the aforementioned VP method.Specifically, panel D shows the results obtained when the sample wasinjected to the loading channel under a pressure of 150 mbar, andthereafter a pressure of 100 mbar, followed by a squeezing voltage (100V), was applied to the separation channel. In the panel D, therespective peaks are as follows: 1: Lysozyme, 2: Trypsin inhibitor, 3:Carbonic anhydrase, 4: Ovalbumin, 5: Serum albumin, 6: Phosphorylase, 7:β-Galactosidase, and 8: Myosin. Panel E shows the results obtained whenthe sample was injected to the loading channel by a pressure of 150mbar, and thereafter a pressure of 150 mbar, followed by a squeezingvoltage (100 V), was applied to the separation channel, according to theaforementioned PP method. In all cases, running voltage was 800 V(electric field of 267 V/cm).

[0199] As a result, protein peaks could not be separated in theconventional microchip electrophoresis, whereas good resolution wasobtained by the aforementioned VP method and PP method.

[0200] According to the PP method shown in panel D, in particular, gooddetection was carried out even when the number of peaks was large,because the resolution is freely variable.

[0201] Furthermore, according to the PP method shown in panel E, 9 to205 kDa proteins could be separated within 15 seconds.

[0202] Additionally, according to the aforementioned VP method and PPmethod, operating time could be shortened as compared to the separationof the same samples by capillary electrophoresis [see FIG. 13].

[0203] For the capillary electrophoresis, the HP^(3D) CE systemmanufactured by Hewlett Packard Company was used. The capillary used wasthe DB-17 capillary of which inner wall was coated with diphenyldimethyl polysiloxane [inner diameter: 0.1 mm, outer diameter: 0.36 mm,total length: 32.5 cm, effective length: 24 cm; manufactured by J&WScientific]. Peptide detection was carried out with absorption at 200 nmas an index. The conditions for the capillary electrophoresis weremethod: CZE, electrode setting: anode-cathode, capillary temperature:25° C., and running voltage: 10 kV.

[0204] (2) Effects on the Interaction of Two Kinds of Proteins in theSeparation Process of Microchip Electrophoresis

[0205] Using bovine insulin (MW: 5733.5) and myogrobin (MW: 16950.9)[both manufactured by Sigma Company], an electric field of 100 to 500 V(loading voltage) was applied to the loading channel under theconditions of no running buffer in the outlet [3 in FIG. 11], to injectthe sample and microchip electrophoresis was conducted, according to theaforementioned VP method. The results are shown in FIG. 14.

[0206] Under these conditions, it is evident that the mobility of eachof bovine insulin and myogrobin in panels A and B is the same as themobility of each of the two kinds of proteins shown in panel C. Thisdemonstrates that no mobility changes due to protein interaction areobserved under the above conditions.

[0207] (3) Migration Time-Shortening Effect of Application of PressurePrior to Electrophoresis in the VP Method

[0208] The effects of pressure (no application of pressure, applicationof lower pressure, application of higher pressure) in the application ofpressure process prior to electrophoresis in the aforementioned VPmethod were examined. The results are shown in FIG. 15.

[0209] Panel A shows the results obtained without application ofpressure as in conventional microchip electrophoresis. Panel B shows theresults obtained with application of lower pressure (50 mbar). Panel Cshows the results obtained with application of higher pressure (150mbar).

[0210] As a result, it was shown that migration time could be shortenedby application of pressure. It was also shown that baseline disturbancewas reduced with application of higher pressure (150 mbar).

[0211] (4) Separation of Two Kinds of Proteins

[0212] Bovine insulin and myosin were electrophoresed by theaforementioned VP method. The results are shown in FIG. 16.

[0213] Panels A and B show the results obtained when bovine insulin andmyosin, respectively, were separated by conventional microchipelectrophoresis. Panel C shows the results obtained when bovine insulinand myosin were separated by the aforementioned VP method.

[0214] Since there are no mobility changes due to protein interaction inthe VP method, it was shown that the VP method could shorten themigration times of the two proteins.

[0215] (5) Polysaccharide Detection

[0216] α-D(+)-Galacturonic acid monohydrate, β-1,3-Glucan (curdlan)[both manufactured by Wako Pure Chemical Industries, Ltd.] andD-Glucuronic acid [manufactured by Nacalai Tesque, Inc.] were used aspolysaccharide standards. The seaweed extract obtained by ProductionExample 1 above was used as the natural sample of polysaccharide. Eachof these polysaccharides was dissolved in deionized water to obtain a 1to 2 M polysaccharide solution.

[0217] The running buffer for polysaccharides used was 0.1 M Tris-boratebuffer (pH 8.5). In the microchip, 1 μl of SYPRO Orange was reacted with20 μl of each polysaccharide solution. Polysaccharides were detected byabsorption at 260 nm or 280 nm.

[0218] Conditions for the detection of the aforementionedpolysaccharides were studied for each of the aforementioned VP methodand PP method. For control, conventional microchip electrophoresis wasconducted to detect polysaccharides. The results are shown in FIG. 17.

[0219] Panel A shows the results obtained when a voltage of 500 V(loading voltage) was applied for 20 seconds in conventional microchipelectrophoresis. Panel B shows the results obtained when a voltage of300 V (loading voltage) was applied for 10 seconds in conventionalmicrochip electrophoresis. As seen from these results, no peaks could bedetected by the conventional method.

[0220] Panel C shows the results obtained when a voltage of 500 V(loading voltage) was applied to the loading channel for 20 secondsunder the conditions of no running buffer in the outlet, to inject thesample and thereafter a low pressure (50 mbar) was applied to theseparation channel, in the VP method.

[0221] Panel D shows the results obtained when a voltage of 300 V(loading voltage) was applied to the loading channel for 20 secondsunder the conditions of no running buffer in the outlet, to inject thesample and thereafter a low pressure (50 mbar) was applied to theseparation channel, in the VP method.

[0222] Panel E shows the results obtained when a voltage of 300 V(loading voltage) was applied to the loading channel for 20 secondsunder the conditions of no running buffer in the outlet, to inject thesample and thereafter a moderate pressure (100 mbar) was applied to theseparation channel, in the VP method.

[0223] Panel F shows the results obtained when a voltage of 300 V(loading voltage) was applied to the loading channel for 20 secondsunder the conditions of no running buffer in the outlet, to inject thesample and thereafter a high pressure (150 mbar) was applied to theseparation channel, in the VP method.

[0224] Panel G shows the results obtained when a pressure (150 mbar) wasapplied to the loading channel under the conditions of no running bufferin the outlet, to inject the sample and thereafter a low pressure (50mbar) was applied to the separation channel, in the PP method.

[0225] Panel H shows the results obtained when a pressure (150 mbar) wasapplied to the loading channel under the conditions of no running bufferin the outlet, to inject the sample and thereafter a high pressure (50mbar) was applied to the separation channel, in the PP method.

[0226] In all cases, running voltage was 800 V (electric field of 267V/cm). As a result, it was shown that migration time could be furthershortened and good resolution could be obtained under the conditions inpanel H.

[0227] (6) DNA Detection

[0228] Solutions of 0.1 μg to 500 μg/μl DNA [ladder of 10 kb in size(manufactured by Funakoshi)] were used as the samples. A solutioncomprising 0.01 to 1.0% by weight methyl cellulose contained in a buffercomprising 10 mM to 0.15 M Tris-borate buffer (pH 7.0 to 10.0) was usedas the running buffer, and microchip electrophoresis was conductedaccording to the VP method and PP method in (5) above.

[0229] As a result, it was shown that migration time was at least 170seconds for 10 kilobases in conventional microchip electrophoresis,whereas migration time could be shortened to about 50 seconds and goodresolution could be obtained according to the VP method and PP method.

[0230] Industrial Applicability

[0231] According to the method of electrophoresis and the method foranalyzing macromolecular compound of the present invention, it ispossible to achieve high resolution quickly. Therefore, the methods areuseful in the High Through-put screening analysis of proteins or sugarchains in gene analysis, proteome analysis or glycome analysis, and areexpected to be applied in medical diagnostic apparatuses and theelucidation of biological functions, mechanisms of onset of diseases,etc.

1. A separation carrier usable for capillary electrophoresis ormicrochip electrophoresis, wherein the separation carrier comprises onekind of compound selected from the group consisting of β-glucan andmethyl cellulose.
 2. The separation carrier according to claim 1,wherein the separation carrier comprises as the β-glucan at least onekind selected from the group consisting of laminaran containingβ-1,3-glucan, curdlan containing β-1,3-glucan, a plant extractcontaining β-1,3-glucan, a seaweed extract containing β-1,3-glucan, anyeast extract containing β-1,3-glucan, a fungal extract containingβ-1,3-glucan, and a fungal cultured medium containing β-1,3-glucan. 3.The separation carrier according to claim 1, wherein the separationcarrier comprises a seaweed extract containing β-1,3-glucan as theβ-glucan.
 4. The separation carrier according to claim 3, wherein theseaweed extract is an extract obtained by subjecting raw materialseaweed to one kind of extraction method selected from the groupconsisting of a water extraction, an acid/alkali extraction and asolvent extraction.
 5. A running buffer usable for capillaryelectrophoresis or microchip electrophoresis, wherein the running buffercomprises the separation carrier of any one of claims 1 to
 4. 6. Therunning buffer according to claim 5, wherein the running buffer is onekind of buffer selected from the group consisting of the following (1)to (3): (1) a buffer comprising a phosphate buffer at pH 1.0 to 12.0 ata concentration of 1 mM to 0.5 M; (2) a buffer comprising a boratebuffer at pH 5.0 to 11.0 at a concentration of 1 mM to 0.5 M; and (3) abuffer comprising a Tris-borate buffer at pH 5.0 to 11.0 at aconcentration of 1 mM to 0.5 M, and wherein the running buffer comprisesmethyl cellulose at a concentration of 0.001 to 0.5% by weight.
 7. Therunning buffer according to claim 5, wherein the running buffer is onekind of buffer selected from the group consisting of the following (1)to (4): (1) a buffer comprising a phosphate buffer at pH 1.0 to 12.0 ata concentration of 1 mM to 0.5 M; (2) a buffer comprising a boratebuffer at pH 5.0 to 11.0 at a concentration of 1 mM to 0.5 M; (3) abuffer comprising a Tris-borate buffer at pH 5.0 to 11.0 at aconcentration of 1 mM to 0.5 M; and (4) a buffer further comprising0.001 to 1.0% by weight of methyl cellulose in the buffer of the aboveitem (3), and wherein the running buffer comprises curdlan at aconcentration of 0.000001 to 0.1% by weight.
 8. The running bufferaccording to claim 5, wherein the running buffer is one kind of bufferselected from the group consisting of the following (1) to (4): (1) abuffer comprising a phosphate buffer at pH 1.0 to 12.0 at aconcentration of 1 mM to 0.5 M; (2) a buffer comprising a borate bufferat pH 5.0 to 11.0 at a concentration of 1 mM to 0.5 M; (3) a buffercomprising a Tris-borate buffer at pH 5.0 to 11.0 at a concentration of1 mM to 0.5 M; and (4) a buffer further comprising 0.001 to 1.0% byweight of methyl cellulose in the buffer of the above item (3), andwherein the running buffer comprises a seaweed extract at aconcentration of 0.000001 to 0.1% by weight.
 9. A method ofelectrophoresis, characterized in that a sample comprisingmacromolecular compounds is run in capillary electrophoresis ormicrochip electrophoresis in the presence of the running buffer of anyone of claims 5 to
 8. 10. The method of electrophoresis according toclaim 9, wherein the macromolecular compounds are one kind selected fromthe group consisting of a protein, a peptide, an amino acid, a sugarchain, a polysaccharide and a nucleic acid.
 11. A method ofelectrophoresis, characterized by comprising the steps of injecting asample comprising macromolecular compounds to a capillary, then applyingpressure thereto, and then running the sample in an electric field forelectrophoresis such that the macromolecular compound can be separated,in capillary electrophoresis.
 12. The method of electrophoresisaccording to claim 11, wherein the macromolecular compounds are one kindselected from the group consisting of a protein, a peptide, an aminoacid, a sugar chain, a polysaccharide and a nucleic acid.
 13. The methodof electrophoresis according to claim 11 or 12, comprising the steps:(a) injecting a sample into a sample injection port of a capillary byapplication of pressure or an electrical injection, wherein thecapillary comprises the sample injection port and an outlet, and thecapillary is filled with a running buffer [hereinafter referred to asstep (a)]; and (b) applying pressure and then running the sample[hereinafter referred to as step (b)] in capillary electrophoresis. 14.The method of electrophoresis according to claim 13, wherein the sampleis injected into the capillary under the conditions of no water or norunning buffer set in the outlet of the capillary in the step (a), andwherein pressure is applied to water or buffer in the step (b).
 15. Themethod of electrophoresis according to claim 13 or 14, wherein thesample is injected into the capillary by an electrical injection at 1 to30 kV for 1 to 30 seconds in the step (a), and wherein the sample is runin an electric field for electrophoresis of 20 V/cm to 10 kV/cm in thestep (b).
 16. The method of electrophoresis according to claim 13 or 14,wherein the sample is injected into the capillary by an electricalinjection at 1 to 30 kV for 1 to 60 seconds in the step (a), and whereina pressure of 2 to 50 mbar is applied for 2 to 30 seconds in the step(b).
 17. The method of electrophoresis according to any one of claim 11or 16, wherein the sample is run in the presence of the running bufferof any one of claims 5 to
 8. 18. A method of electrophoresis,characterized by comprising the steps: (A) using a microchip comprisinga loading channel, a separation channel crossing the loading channel, asample reservoir arranged at one end of the loading channel, and anoutlet arranged at the other end of the loading channel, wherein theloading channel and the separation channel are filled with a runningbuffer, applying voltage or pressure to the loading channel, to supply asample comprising macromolecular compounds from the sample reservoir,thereby introducing the sample into the separation channel [hereinafterreferred to as step (A)]; and (B) applying pressure to the separationchannel, and then running the sample [hereinafter referred to as step(B)].
 19. The method of electrophoresis according to claim 18, whereinthe macromolecular compounds are one kind selected from the groupconsisting of a protein, a peptide, an amino acid, a sugar chain, apolysaccharide and a nucleic acid.
 20. The method of electrophoresisaccording to claim 18 or 19, wherein resolution is adjusted bycontrolling a degree of pressure applied in the step (B).
 21. The methodof electrophoresis according to any one of claims 18 to 20, wherein thesample is introduced into the separation channel by applying voltage tothe loading channel under the conditions of no running buffer set in theoutlet in the step (A); and wherein voltage is applied to the loadingchannel and the separation channel, thereby running the sample, in thestep (B).
 22. The method of electrophoresis according to claim 21,wherein a voltage of 10 to 500 V (loading voltage) is applied to theloading channel for 2 to 60 seconds in the step (A); and wherein avoltage of 10 to 500 V (squeezing voltage) is applied to the loadingchannel and, an electric field of 20 V/cm to 50 kV/cm is applied to theseparation channel in the step (B).
 23. The method of electrophoresisaccording to any one of claims 18 to 20, wherein the sample isintroduced into the separation channel by applying pressure to thesample reservoir under the conditions of no running buffer set in theoutlet in the step (A); and wherein pressure is applied to theseparation channel and then the sample is run in step the (B).
 24. Themethod of electrophoresis according to claim 23, wherein a pressure of 1to 1520 mbar is applied to the sample reservoir in the step (A); andwherein a pressure of 1 to 1520 mbar is applied to the separationchannel, and then an electric field of 20 V/cm to 50 kV/cm is appliedthereto, in step the (B).
 25. The method of electrophoresis according toany one of claims 18 to 24, wherein proteins having molecular weights of9 to 205 kDa are separated within 15 seconds.
 26. The method ofelectrophoresis according to any one of claims 18 to 24, wherein sugarscomprising 2 to 100 monosaccharides as a constitutive sacccharide areseparated within 15 seconds.
 27. The method of electrophoresis accordingto any one of claims 18 to 24, wherein nucleic acids of 10 bases to 10kilobases are separated within 50 seconds.
 28. A method for analyzingmacromolecular compounds, characterized by comprising the steps ofrunning a sample comprising macromolecular compounds by the method ofelectrophoresis of any one of claims 9 to 27, thereby separating themacromolecular compounds; and measuring mobility by detecting theseparated macromolecular compounds.
 29. The method for analyzingmacromolecular compounds according to claim 28, wherein themacromolecular compounds are one kind selected from the group consistingof a protein, a peptide, an amino acid, a sugar chain, a polysaccharideand a nucleic acid.
 30. The method for analyzing macromolecularcompounds according to claim 28 or 29, wherein the separatedmacromolecular compounds are detected by at least one means selectedfrom the group consisting of a determination of UV wavelength lightabsorption, a fluorescence detection, an electrochemical detection and achemiluminescence detection.